The capture and isolation of biological targets (pathogens, bacteria, cells, functionalized micro-beads, etc.) are critically important in many clinical diagnostic, screening, environmental assessment and quality control applications. For many of these applications, there is a need for rapid and low-cost detection/identification assays. In the area of food safety, foodborne disease is a serious public health threat and thus rapid detection of potentially life-threatening pathogens remains a major public health challenge (Yang 2006). Similar challenges are found in the field of clinical diagnostics where rapid detection of pathogens in a patient's blood is sought; and in environmental and biosecurity applications where identification of bacteria and other contaminants from water samples are desired. Over the past several years, a variety of methods have been investigated for the detection of bacteria and other biological targets in food or water, for example, immunological assays (Koubová 2001; Vaughan 2001; Sewell 2003), nucleic acid-based tests (Ingianni 2001; Choi 2002; Amagliani 2004) and physicochemical tests based on bacterial growth (Wawerla 1999; Firstenberg-Eden 2000).
Among the above mentioned methods, immuno-capture based assays are of great interest due to the high sensitivity and specificity of antigen-antibody immuno-interaction. Antigens present on surfaces of species/objects of interest (pathogens, bacteria, microbeads) suspended in a biological fluid/sample are captured by specific antibodies immobilized on to a surface. While the antigen-antibody interaction have been primarily used in the immuno-capture assays, various techniques can replace this interaction with other moieties such as aptamers (peptides/oligonucleotide sequences) and biophages that are thought be provide better capture (Zourob 2008). In such assays, the probability of capture of the targets is directly related to the velocity of the fluid above the functionalized surface (antibody, biophage, aptamer-coated surface), with higher probability of capture being obtained at lower flow rates. The order of magnitude of the liquid velocity at which reasonable values for probability of capture are obtained ranges in the tens of micrometers per second. At these relatively slow flow rates and with the typical sample volumes in use in many biological protocols (milliliter to hundreds of microliters), an assay or analysis can take significant time, thus preventing rapid detection.
The main reason these extremely low flow rates are used in immuno and other capture assays originates in the hydrodynamic interaction of species or objects with the functionalized surface. Particles flowing near a rigid surface undergo a “wall effect” where an asymmetric wake of the particles near the surface leads to lift forces away from the surface (Zeng 2005). Thus, the “natural” tendency of functionalized rigid surfaces is to repel particles flowing near the surface, the repelling force being higher at higher velocities of the particles. Consequently, the velocity of the liquid must be as small as possible in order to allow particles to attach to the functionalized surfaces. The forces that naturally push the particles against the capture area of the surface are thermal, gravitational and diffusive effects in the biological liquid sample.
In order to increase the efficiency of species binding to functionalized capture sites, several methods have been proposed. One of them employs an array of interdigitated metallic electrodes and the dielectrophoretic force to give pathogens an additional push against the capture sites (Li 2002; Yang 2006). The dielectrophoretic force acting on pathogens originates in the ability of the pathogens to polarize in the presence of electric fields. This force can be adjusted by tuning the amplitude and frequency of the applied AC fields. An equivalent method employs electromagnetic cellular polarization and optical scattering for direct detection but without the use of any biochemical marker (Choi 2006).
One and the most important drawback of dielectrophoresis-based capture approaches is related to the short range action of the dielectrophoretic force itself, which, in practical microfluidic applications is only on the order of tenths of micrometers (Li 2002). This limits the size of the microfluidic channels, thus the overall throughput of the device. Moreover, the use of complicated arrays of electrodes increases the number of fabrication steps (thus the cost per unit device) associated with the electronics needed to generate the necessary high frequency AC voltages. This is detrimental when single-use, low-cost and portable devices for point-of-care applications are intended.
Another approach is based on immuno-magnetic capture and separation (Dwivedi 2011). Instead of forcing particles to bind to rigid (fixed) walls, the antibodies are deposited onto the surface of superparamagnetic beads. These beads become magnetic only in the presence of external magnetic fields and return immediately to the non-magnetized state as the magnetic field is removed. This is an important property for immuno-magnetic capture since the beads will freely interact with the target antigens (pathogens) in stagnant liquid suspensions without clustering together by mutual magnetic interactions. The process of capture can be slightly accelerated if moderate vortexing (agitation) of liquid suspensions is induced. Commercial devices, such as the well known BeadRetriever™ from Dynal Biotech Ltd. (Wirral, UK) based on the inverse magnetic particle processing principle, are able to reduce the capture time further by moving the particles along small tubes containing the sample with the aid of a magnetic bar. Related methods further decrease the detection time by adding features such as quantum dots for enhanced fluorescence (Su 2004), magnetic relaxation (Kaittanis 2007) and time-of-flight mass spectrometry (Madonna 2001).
In immuno-magnetic capture using superparamagnetic beads, the time needed by functionalized beads to bind to specific pathogens present in the sample may be lowered by stirring the solution to increase the probability of capture. However, the stirring speed is limited to the same fluid-to-solid relative velocities as in the static case, mainly due to the same hydrodynamic wall effect that manifests at the surface of moving beads. Consequently, the fundamental problem related to the wall effects that repel particles from functionalized surfaces is not addressed.
Immuno-magnetic capture using superparamagnetic beads may be implemented in microfluidic devices (e.g. Lee 2010; Lee 2011). The beads are used as a carrier surface for the capture of a target molecule. In these cases, centrifugal force generated by the rotating device is used to pump fluids through the device and to move the beads from chamber to chamber. Centrifugal force is not used to directionally immobilize target particles on to an immobile capture surface.
There remains a need for increasing capture efficiency of a target molecule in a capture assay in a microfluidic device.